Download development in gagea lutea (l.) ker.-gaw.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 89–96, 2005
DISTRIBUTION OF MICROTUBULES DURING REGULAR
AND DISTURBED MICROSPOROGENESIS AND POLLEN GRAIN
DEVELOPMENT IN GAGEA LUTEA (L.) KER.-GAW.
JERZY BOHDANOWICZ*1, EWA SZCZUKA**2, JOANNA ŚWIERCZYŃSKA1, JOLANTA SOBIESKA3,
AND MARIA KOŚCIŃSKA-PAJA˛K4
1
Department of Genetics and Cytology, University of Gdańsk,
ul. Kładki 24, 80–822 Gdańsk, Poland
2
Department of Plant Anatomy and Cytology, Maria-Curie Skłodowska University,
ul. Akademicka 19, 20–033 Lublin, Poland,
3
Department of Biology and Horticulture, University College of Arts and Natural Science,
ul. Krakowska 26, 27–600 Sandomierz, Poland
4
Department of Plant Cytology and Embryology, Jagiellonian University,
ul. Grodzka 52, 31–044 Cracow, Poland
Received January 20, 2005; revision accepted June 30, 2005
The microtubular cytoskeleton in dividing microsporocytes and developing pollen grains of Gagea lutea (L.) Ker.-Gaw.
(Liliaceae) was investigated with a modified indirect immunofluorescence method. Meiotic and mitotic stages were
identified by DAPI staining. The microtubular cytoskeleton was compared in plants originating from natural localities
and others grown in the laboratory. In natural conditions, microsporocytes and pollen grains of wild early-spring
Gagea lutea plants are subjected to abiotic factors including cold exposure and lack of water. The persistent influence
of these factors can disturb microtubular cytoskeleton functioning. The following disturbances were observed in the
course of microsporogenesis and pollen development: abnormal chromosome configurations in the metaphase of
meiosis I; abnormally divided dyads with irregular, radial microtubule systems around the nuclei; the formation of
differently sized microspores with irregular shapes, and irregular division; and the formation of pollen grains with
vacuoles abnormal for their development stage. Similar kinds of disturbances were observed after 1.5 months of cold
treatment (4˚C) and drying in the laboratory. These abiotic factors simulated in laboratory conditions caused more
disturbances in the course of microsporogenesis and produced more frequent defective pollen grains than in the sample
that had experienced cold and drying in natural conditions.
Key words: Gagea lutea, microtubular cytoskeleton, microsporogenesis, disturbances, abiotic
factors.
INTRODUCTION
Microtubules are major components of all eukaryotic
cells, playing a basic role in processes such as chromosome segregation, cellulose deposition, vesicle transport, cell motility, maintenance of cell shape, and cell
expansion (Waldin et al., 1992). Because of their great
importance in the cell, microtubules have been studied
intensively by different methods (for references, see:
Vesk et al., 1996). The division of meiotic cells in plants
involves microtubule configurations, most of which are
similar to those present in somatic tissues. Three
microtubular configurations occur in meiotic plant cells
during microsporogenesis: the microtubular system in
the cortical cytoplasm and at the nuclear envelope;
metaphase I and II spindles; and phragmoplasts after
the first and second meiotic divisions. These configurations have been the subject of many detailed investigations (van Lammeren et al., 1985; Hogan, 1987; Brown
and Lemmon, 1988b, 1991a, 1996, 2000; Traas et al.,
1989; Liu et al., 1993; Genualdo et al., 1998; Giełwanowska et al., 2003, 2005). They appear regularly during
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PL ISSN 0001-5296
© Polish Academy of Sciences, Cracow 2005
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Bohdanowicz et al.
typical meiosis completed with successive as well as
simultaneous cytokinesis.
In the meiocytes of the successive type of cytokinesis (sporo- and microsporocytes), microtubules forming the phragmoplast develop in the equatorial
region following meiosis I; in the middle plane, the cell
plate is set up to divide the binucleate meiocyte (Brown
and Lemmon, 199la). After meiosis, the final quadripartitioning into four microspores takes place with the
formation of microtubule arrays that interconnect the
nuclei in both halves of the dyad. In general, the planes
of division in micro- and sporogenesis depend on the
position of the nuclei, which define cytoplasmic domains via a radial system of microtubules (Brown and
Lemmon, 1988a,b, 1991a,b, 1996). The organization of
microtubules during pollen grain development resembles the organization found during mitotic division,
although the division into a two-cellular pollen grain is
asymmetric.
Microsporogenesis and pollen grain development
have been at the center of interest of biologists because
generative reproduction in plants depends on proper
pollen structure and function. Proper organization of
the microtubular cytoskeleton during meiotic and mitotic divisions is essential for the formation of highquality, viable pollen grains. Disturbances during both
processes decrease the chance of effective pollination
and fertilization of maternal plants.
In this paper we report on the distribution of
microtubules (microtubular cytoskeleton elements)
formed during regular microsporogenesis and pollen
grain development in Gagea lutea, and describe disturbances in the course of both these processes. We also
discuss the influence of abiotic factors such as variable
temperature (cold treatment) and periodic lack of
water on the microtubular cytoskeleton during these
processes, which are so significant for plant reproduction.
MATERIALS AND METHODS
PLANT MATERIAL
Plants of Gagea lutea (L.) Ker.-Gaw. (Liliaceae) growing in natural habitats in Gdańsk and Stalowa Wola
were used in the study. Flower buds were collected in
winter months (December and January), and 75 G.
lutea plants were transplanted to pots with soil and
transported to the laboratory. These plants were kept
refrigerated at 4˚C and not watered for 1.5 months
during the winter. After that period of cold and drying,
the plants were cultivated in laboratory conditions at
room temperature.
IMMUNOLOCALIZATION OF β-TUBULIN
IN SECTIONED MATERIAL
Anthers were excised from flower buds and fixed immediately in 4% formaldehyde (freshly prepared from
paraformaldehyde) and 0.25% glutaraldehyde in
microtubule stabilizing buffer (MSB) for 4 h at room
temperature. The MSB consisted of 50 mM PIPES
(piperazine-N,N’-bis[2-ethanesulfonic acid]), 10 mM
EGTA (ethylene glycol-bis[β-aminoethyl ether]
N,N,N’,N’-tetraacetic acid), and 1 mM MgCl2, pH 6.8.
The fixed anthers were processed according to the
procedure of Vitha et al. (1997, 2000). After fixation
and three rinses in MSB, they were dehydrated in a
graded ethanol series containing 10 mM dithiothreitol
(DTT, Sigma) (Brown et al., 1989) to minimize the
background of cytoplasm. Then the plant material was
infiltrated with Steedman’s Wax: polyethylene glycol
400 distearate (Aldrich) and cetyl alcohol (Sigma) in a
9:1 (w/w) proportion. After polymerization of the wax,
10 μm sections were made from the embedded anthers
and stretched on a small drop of distilled water on
microscope slides coated with Mayer’s egg albumen.
The sections were dried overnight, dewaxed in ethanol,
rehydrated in an ethanol-PBS series, washed in PBS
Figs. 1–20. Regular microsporogenesis and pollen grain development in Gagea lutea. Microtubules visualized by indirect
immunofluorescence with a fluorescence microscope. Figs. 1, 2. Matching pair of prophase I microsporocytes. Fig. 1. Arrays
of microtubules in the cytoplasm. Fig. 2. Nucleus (N) after DAPI staining. Figs. 3, 4. Metaphase I. Fig. 3. Short kinetochore
(arrowheads) and thinner, longer interzonal microtubules (arrow) in the bipolar spindle (asterisks). Fig. 4. Compact
chromosome plate after DAPI staining. Figs. 5, 6. Matching pair of telophase I microsporocytes. Fig. 5. Microtubules of a
phragmoplast (arrows), with system of radial microtubules (arrowheads) around the nucleus. Fig. 6. Two flattened nuclei (N)
stained with DAPI. Figs. 7, 8. Dyad. Fig. 7. System of radial microtubules (arrowheads) around the nuclei: at left, narrow
part of phragmoplast (arrow). Fig. 8. Nuclei (N) after DAPI staining. Figs. 9, 10. Tetrad of microspores. Fig. 9. System of
radial microtubules inside each of four microspores (arrowheads in one of the cells). Fig. 10. Four nuclei (N) of the microspores
stained with DAPI. Figs. 11, 12. Matching pair of released microspores. Fig. 11. Abundant microtubular network extends
through the cytoplasm. Fig. 12. Nucleus (N) after DAPI staining. Figs. 13, 14. Matching pair of dividing pollen grains. Fig. 13.
Microtubular phragmoplast between generative (GN) and vegetative (VN) nuclei, which are visible after DAPI staining in
Fig. 14. Arrows point to the dark line of the new cell plate forming between the vegetative and generative cells. Figs. 15, 16.
Two-cellular pollen grain. Fig. 15. Dense layer of thick microtubules gathered in generative cell (arrows): a subtle microtubular
net is visible in the cytoplasm of the vegetative cell. Fig. 16. Vegetative (VN) and generative (GN) nuclei after DAPI staining.
Figs. 17, 18. Two-cellular pollen grain older than in Fig. 15. Fig. 17. Thick strings of microtubules in generative cell. Fig. 18.
Generative nucleus (GN) after DAPI staining (vegetative nucleus is present in another section). Figs. 19, 20. Mature pollen
grain. Fig. 19. Microtubules gathered in generative cell. Fig. 20. Vegetative (VN) and generative (GN) nuclei of the pollen
grain shown in Fig. 19.
Microtubules in pollen meiosis of Gagea lutea (L.) Ker.Gaw.
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and preincubated in PBS with 0.1% BSA for 30 min.
The samples were incubated overnight at 4˚C with
mouse anti-β-tubulin monoclonal antibody (Amersham) diluted 1:200 in PBS. Then the sections were
rinsed three times in PBS and incubated for 4 h in
secondary Alexa 488-conjugated anti-mouse antibody
(Molecular Probes) diluted 1:800 in PBS. The slides
were rinsed in PBS, and the nuclei stained with 1 μg/ml
4’,6’-diamidino–2-phenylindole dihydrochloride
(DAPI) in PBS. Then the sections were treated with
0.01% toluidine blue to diminish cell wall autofluorescence and mounted in antifading solution (Citifluor,
Agar). In control experiments conducted in a similar
manner and omitting the first antibody, no tubulin
staining was detected.
FLUORESCENCE MICROSCOPY
Fluorescence was observed with a Nikon Eclipse E 800
epifluorescence microscope, using a B–1E filter (EX
470–490 nm, DM 505, BA 520–560) to observe tubulin
labeled with Alexa 488, and a UV–2A filter (EX 330–
380, DM 400, BA 420) to examine DAPI-stained nuclei.
Photographs were taken on Kodak T-max film, ASA
400. Image processing was done with Adobe Photoshop.
LIGHT MICROSCOPY
Acetocarmine stain was used for visualization of chromosomes. Alexander’s method was applied to differentiate fertile and sterile pollen grains (Dafni, 1992).
Sterile pollen grains were counted with a hemacytometer.
RESULTS
In Gagea lutea plants growing in natural conditions,
70% of the pollen grains underwent normal microsporogenesis and pollen grain development. Meiosis begins in September, but we observed the meiocytes at
different stages of development during autumn and
winter months. At prophase of meiosis I, microtubules
are dispersed throughout the cytoplasm of the microsporocyte. At the stage when the meiotic cell is highly
polarized and the nucleus occupies one of its poles, the
microtubular network is distributed asymmetrically
(Figs. 1, 2). The microtubules form arrays scattered
randomly in the cytoplasm, but near the nuclear envelope they gather in a thin, compact layer. Such a
layer only occurs close to the nuclear envelope adjacent
to the cytoplasmic region of the microsporocyte. During
the metaphase of meiosis I, a short kinetochore and
thinner, longer interzonal microtubules form the bipolar spindle (Fig. 3), which acts during anaphase. The
compact chromosome plate occupies the equatorial
plane of the microsporocyte (Fig. 4). At the end of
meiosis I, the arrays of microtubules extend into the
internuclear region from the sister nuclei, and a conspicuous phragmoplast is formed (Fig. 5) between the
telophase nuclei (Fig. 6). The phragmoplast disappears
after the cell plate dividing the meiocyte into two
separate cells is established. Following microsporogenesis, microtubules appear at the nuclear envelopes of
the dyad (Figs. 7, 8) and, starting from prophase of
meiosis II, they behave in the same way as in the
successive stages of meiosis I. Meiosis II is completed
with the formation of a microspore tetrad (Figs. 9, 10).
In the separated young microspores a system of radial
microtubules develops around each of the nuclei.
The young released microspore is spherical; later
its shape changes. Most of its volume is occupied by
cytoplasm in which the clear microtubule arrays form
a net (Fig. 11). The nucleus of the microspore is in a
peripheral position close to the cell wall (Fig. 12). In
this asymmetrical position the microspore nucleus
divides mitotically. At the end of the mitosis a conspicuous phragmoplast is set up between the generative and
vegetative nuclei (Figs. 13, 14). The central part of the
phragmoplast disintegrates, and the cell wall appears
in place of the former phragmoplast, dividing the pollen
grain into two asymmetric cells. In the two-cellular
pollen grain, microtubules are observed in abundance
in the generative cell, whereas in the cytoplasm of the
vegetative cell a subtle microtubular net is visible (Fig.
15). At this stage, the generative and vegetative nuclei
Figs. 21–33. Disturbances in the course of microsporogenesis and pollen grain development in Gagea lutea. Microtubules
visualized by indirect immunofluorescence. Figs. 21–23. Metaphase I microsporocyte. Fig. 21. Irregular spindle. Fig. 22.
Chromosomes (asterisks; visible after DAPI staining) scattered irregularly in the cytoplasm. Fig. 23. Chromosomes stained
with acetocarmine, one outside the chromosome plate (arrow). Figs. 24–27. Dyad. Fig. 24. Differently sized cells with systems
of radiating microtubules. Fig. 25. Irregular-shaped nuclei (N) and micronucleus in one of the dyad cells (arrow). Fig. 26.
Irregularly distributed microtubules in the radial systems around the dyad nuclei, and incomplete cell wall between two
post-telophase I nuclei (arrowheads). Fig. 27. Dyad nuclei (N) after DAPI staining, with remnants of chromosome bridge in
the central part of the microsporocyte (arrow). Figs. 28, 29. Three microspores of the tetrad. Fig. 28. Mitotic spindles in the
microspores. Fig. 29. Chromosome plates visible after DAPI staining. Figs. 30, 31. Matching pair of two-cellular pollen grains.
Fig. 30. Extended microtubular system and two vacuoles (V) in generative cell cytoplasm. Fig. 31. Vegetative (VN) and
generative (GN) nuclei after DAPI staining. Fig. 32. Two-cellular pollen grain older than in Fig. 31 after DAPI staining, with
vegetative (VN) and generative (GN) nuclei visible, and three micronuclei (arrows). Fig. 33. Mature pollen grains stained by
Alexander’s method. Non-aborted pollen grains are dark, and aborted ones light (arrows).
Microtubules in pollen meiosis of Gagea lutea (L.) Ker.Gaw.
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are the same size, although the DNA of the generative
cell gives stronger fluorescence after DAPI staining
(Fig. 16). In the further course of pollen grain development, the generative cell moves from the periphery
towards the pollen grain center. At this stage, occasionally thick and long arrays of microtubules form a
spindle-shaped structure inside the generative cell: in
the cytoplasm of the vegetative cell a subtle microtubular net is barely visible (Figs. 17, 18). In the mature
pollen grain, usually the generative cell is almost
spherical, with an extensive system of cortical microtubules (Fig. 19). The microtubules are gathered
mainly in the generative cell. The vegetative nucleus
is smaller than the generative one (Fig. 20).
In the anthers of plants growing in natural conditions, up to 30% of the pollen grains develop abnormally
as the viable pollen grains form. The disturbances can
occur in the course of microsporogenesis or pollen grain
development. The first disturbance observed was at
metaphase of meiosis I. Figure 21 shows an atypical
meiotic spindle with scattered microtubule bundles.
Instead of fusiform shape, the spindle assumes an
irregular form without typical poles. In a microsporocyte with such a spindle, the chromosomes do not form
a regular metaphase plate (Fig. 22). Chromosomes can
occur outside the metaphase plate (Fig. 23), resulting
in the appearance of micronuclei during further stages
of microsporogenesis (Figs. 24, 25).
After the first meiotic division, irregularly distributed microtubules were observed in radial systems
around the dyad nuclei (Fig. 26). In such a dyad, the
cell wall between two post-telophase I nuclei is incomplete, and there are remnants of chromosome bridges
between the nuclei (Fig. 27).
Pollen mitosis can occur in tetrad microspores. All
four microspores in a tetrad can divide synchronously
(Figs. 28, 29), but sometimes four different mitotic
stages can be found in one tetrad. The newly formed
microspores in one tetrad can be irregular in shape and
can differ in size.
In the young, two-cellular pollen grain, thick arrays of microtubules surround the generative nucleus.
Large vacuoles are formed in the peripheral parts of
the pollen grain (Fig. 30). The aberrant size of these
vacuoles is shown in Figure 31.
The cytoskeleton disturbances described above influence the structure of the mature pollen grain. Micronuclei were observed in some pollen grains (Fig. 32),
varying in number from one to seven. In most cases,
the disturbances during microsporogenesis and pollen
grain development lead to the production of a sterile
pollen grain (Fig. 33).
In the distribution of microtubules during both
regular and disturbed microsporogenesis and pollen
grain development, there were no observed differences
between Gagea lutea plants originating from natural
habitats in Gdańsk and Stalowa Wola.
The same kinds of disturbances as described above
were also observed after 1.5 months of cold (4˚C) and
drying in laboratory conditions. Among the pollen
grains developing in laboratory conditions, 50% were
aborted. The remaining pollen grains had undisturbed
development.
DISCUSSION
Two well-known processes typical for the majority of
angiosperms lead to the formation of mature pollen
grains: microsporogenesis and pollen grain development. Both of them also lead to the production of
mature pollen grains in G. lutea. In this species, microsporogenesis is accomplished by successive cytokinesis.
Such a type of cytokinesis, with cell walls assembled
after both telophase I and telophase II, is typical for
most Monocotyledones, Bryophyta, Pteridophyta and
Gymnospermae. In G. lutea growing in natural conditions, in 70% of the cases there are no disturbances of
these two processes – successive meiotic division and
pollen grain development. In both microsporogenesis
and pollen development, the microtubular cytoskeleton
plays a fundamental role in forming spindles, phragmoplasts and microtubular networks in the cortical
cytoplasm and at the surface of the nuclear envelope
(Hogan, 1987; Busby and Gunning, 1988; Brown and
Lemmon, 1988b, 1991a, 1996, 2000; van Lammeren et
al., 1985; Staiger and Cande, 1990, 1991; Staiger and
Lloyd, 1991; Liu et al., 1993; Genualdo et al., 1998;
Shimamura et al., 1998; Giełwanowska et al., 2003).
The changes in the microtubular cytoskeleton during
the regular meiotic and mitotic divisions leading to
two-cellular pollen grains in G. lutea are typical for
most plants and do not require discussion here (for
references, see: Otegui and Staehelin, 2000).
Thirty percent of pollen grains in the G. lutea
anther are sterile. The disturbances leading to this
phenomenon appeared during microsporogenesis and
pollen grain development. The following kinds of disturbances in the course of microsporogenesis were observed: abnormal chromosome configurations in the
metaphase of meiosis I; abnormally divided dyads with
irregular, radial microtubule systems around the nuclei; and the formation of microspores irregular in
shape and differing in size; and irregularity of division.
Vacuoles appeared during the formation of the pollen
grain, but that is abnormal for this developmental
stage. Undoubtedly the observed disturbances are directly linked to the functioning of the microtubular
cytoskeleton. In undisturbed divisions, the configurations of the microtubular cytoskeleton rearrange according to each phase of the plant cell meiotic and
mitotic cycle (Asada and Collins, 1997). Microtubules
forming the cytoskeleton are very dynamic polymers of
α- and β-tubulin heterodimers, and they operate in all
Microtubules in pollen meiosis of Gagea lutea (L.) Ker.Gaw.
key developmental events, including cell division,
growth and differentiation (Marc, 1997). Disturbances
affecting the cytoskeleton might result in disturbed
meiotic or mitotic divisions and abnormal chromosome
segregation.
Gagea lutea is an early-spring monocotyledonous
plant. In Poland’s climate, dividing microsporocytes
and developing pollen grains of wild early-spring
plants are subjected to abiotic factors in winter, which
is the time of microsporogenesis and pollen grain development in G. lutea. Among these factors are variable
temperature (cold exposure) and periodic lack of water.
Both stresses act simultaneously. The persistent influence of these factors, which are natural stresses, can
bring about disturbances in microtubular cytoskeleton
functioning. It is believed that depolymerization of
microtubules induced by lowering temperature causes
the loss of subunits or small fragments from their ends
(for references, see: Schliwa, 1986). If microtubules are
depolymerized in key configurations during microsporogenesis or pollen grain development, both processes
can be disturbed in ways observed in G. lutea. Depending on its intensity and duration, low temperature
generally impairs the metabolic activity, growth and
viability of plant cells. Biomembranes usually become
more rigid at low temperatures, and more energy is
required to activate biochemical processes. All this can
disturb both meiotic and mitotic divisions. On the other
hand, the influence of an abiotic factor like low temperature cannot be judged without considering periodic
lack of water. At low temperatures, plants cannot take
up enough water to cover their requirements. The
strain imposed on the water balance by winter conditions may cause damage due to desiccation as well as
to disturbed meiosis and mitosis (for references, see:
Larcher, 2003).
Disturbances similar to those in the anther of
wild-growing G. lutea were observed in plants exposed
to 1.5 months of cold (4˚C) and drying in the laboratory.
They had more disturbances in the course of microsporogenesis and more defective pollen grains than plants
exposed to those abiotic factors in natural conditions.
The plants in the laboratory probably were subjected
to a more restrictive regime than those growing in the
natural environment.
Proper pollen grain development depends not only
on microtubular cytoskeleton functioning. For
example, in many plant species, male sterility has been
found to be caused by distortions in tapetum and endothecium functioning, or reduction of anthers. Additionally, male sterility can be governed in genetic,
cytoplasmic, or cytoplasmic-genetic ways (Klein, 2000).
The development of the male gametophyte can be disturbed at all steps. These points help explain the high
percentage of abnormal pollen grains (50%) derived in
laboratory conditions. In an investigation of G. lutea
pollen grain germination, Zhang et al. (1995) found
95
that only about 30% of pollen grains were viable and
germinated in the pollen tubes successfully; this low
germination percentage was not unexpected, since a
large amount (even more than 50%) of abnormal,
shrunken pollen grains were present in the samples
they investigated.
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